Abstract
Physical cues such as light, heat, or an electrical field can be utilized for traceless, on-demand activation of the expression of a desired therapeutic gene in appropriately engineered cells with excellent spatiotemporal resolution, good inducibility, and simple reversibility. A similar approach can be applied to build a depolarization-based protein secretion system that enables rapid release of a therapeutic protein pre-stored in intracellular vesicles in mammalian cells. Here, we present a protocol to create designer β-cells that exhibit light-controllable rapid release (within 15 min) of a pre-synthesized proinsulin–nanoluciferase construct from vesicular stores. The construct is cleaved extracellularly to afford secreted insulin as a therapeutic protein and nanoluciferase as a reporter molecule. Such posttranslational remote control offers a much faster response than expression-based systems.
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Acknowledgments
Work in the laboratory of M.F. is financially supported in part through a European Research Council advanced grant (ElectroGene, no. 785800) and in part by the National Centre of Competence in Research (NCCR) for Molecular Systems Engineering as well as the EC Horizon 2020 Framework Programme ENLIGHT.
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Mansouri, M., Fussenegger, M. (2024). Posttranslational Remote Control Mediated by Physical Inducers for Rapid Protein Release in Engineered Mammalian Cells. In: Ceroni, F., Polizzi, K. (eds) Mammalian Synthetic Systems. Methods in Molecular Biology, vol 2774. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3718-0_15
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DOI: https://doi.org/10.1007/978-1-0716-3718-0_15
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